Ecology of the Threatened Thick-Shelled River Mussel Unio Crassus (Philipsson 1788) with Focus on Mussel-Host Interactions

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Ecology of the threatened thick-shelled river mussel Unio crassus (Philipsson 1788) with focus on mussel-host interactions

Lea Dominique Schneider Department of Biology

Introductory paper No. 11 2014

Ecology of the threatened thick-shelled river mussel Unio crassus (Philipsson 1788) with focus on mussel-host interactions

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Lea D. Schneider Department of Biology Karlstad University

Introductory paper No. 11

2014

Contents

1. Introduction ...... 3 2. Freshwater mussel ecology – the order Unionoida ...... 5 2.1. Sexual strategies and larval development...... 5 2.2. The mussel – host-fish relationship ...... 8 3. Status and threats to freshwater bivalves...... 14 4. Conservation strategies ...... 16 5. My doctoral research ...... 17 6. Acknowledgements ...... 19 7. References ...... 20

1. Introduction

Freshwater mussels (phylum Mollusca, class Bivalvia) derive from marine species and inhabit a variety of inland waters such as rivers, streams, lakes and ponds worldwide, with the exception of Antarctica. Generally, the most diverse mussel fauna can be found in North America, holding one third of the world’s mussel species (Strayer 2008). Repeated invasions of marine mussels to freshwaters have led to phylogenetically independent bivalve lineages (Graf 2013), resulting in co-existence of non-related freshwater mussel groups such as cyrenids (formerly the corbiculids, Bieler et al. 2010), dreissenids and unionoids (Watters 2001). Mussel colonization of different environments has induced a multitude of mussel life history traits and physiological adaptations to various habitat conditions. The mussel communities in large rivers, for example, are mainly represented by a variety of generalist species that show similar patterns of adaptation to local conditions. For instance, taxa in large rivers with hard sediments often have sculptured shells which facilitate burrowing, while taxa living in soft substrates produce thinner and flattened shells. On the other hand, mussels in headwater streams often have streamlined, unsculptured shells and are usually highly habitat specific (Bauer 2001c). In addition, mussel diversity increases downstream in watersheds, and beta diversity is greatest between headwater streams (Haag and Warren 1998).

Mussels are not only affected by habitats, but can also substantially alter the habitats they live in. Mussels bioturbate sediments through pedal (foot) movement (Limm and Power 2011) and together with biodeposition of faeces and pseudofaeces (biodeposits) affect nutrient dynamics and availability in freshwaters (Pusch et al. 2001). Moreover, mussel shells increase pore space in the sediment, contributing to interstitial oxygenation as well as sediment stabilisation. As freshwater mussels are filter feeders, they can improve water clarity, particularly when mussels occur in high densities (Strayer et al. 1994). Furthermore, epiphytic and epizoic organisms use mussel shells as habitat (Vaughn and Hakenkamp 2001). Consequently, the abundance of mussels can greatly contribute to ecosystem functioning and freshwater biodiversity (e.g. Aldridge et al. 2007, Vaughn 2010). The high diversity of life history traits among mussels includes some specialised features. For instance, freshwater mussels belonging to the order Unionoida have a long and complex life cycle (usually > 10 years, Israel 1913), where most species have a larval stage that is dependent on a fish host (Barnhart et al. 2008). The unionoid larvae attach to the gills or skin of the fish, on which they parasitize, from a few days to around one year. After they metamorphose to juvenile mussels they fall off the fish and sink to the sediment. They then bury themselves into the sediment, completing development to reproducing adults within a few to approximately 15-20 years (Fig 1). Such specialized life cycles can, although adaptive, face a number of challenges, as both fish community structure and habitat conditions must meet the needs of each life-cycle stage of the mussels (Strayer et al. 2004, Vaughn and Taylor 2000, Österling et al. 2008).

Fig. 1 Life cycle of Unio crassus with the minnow (Phoxinus phoxinus) acting as a host fish for the mussel larvae (glochidia). © UC4LIFE

The freshwater unionid bivalves are one of the most threatened groups of organisms on earth. The global decline of these mussel species is probably related to both anthropogenic activities and the complex life history traits of these mussels (Bogan 2008). For some species, every life stage is negatively affected (Österling 2014). A lack of suitable fish hosts can impair mussel recruitment, affecting population density, potentially leading to extinction (Zale and Neves 1982, Arvidsson et al. 2012). Fish composition has been largely affected by anthropogenic habitat alteration since the industrial revolution. Freshwater mussels are also sensitive to habitat modification, water pollution, land-use change, exotic species introductions and overharvesting (Zahner-Meike and Hanson 2001, Bauer and Wächtler 2001, Strayer et al. 2004, Österling et al. 2008, Österling et al. 2010).

Freshwater mussels have been described as keystone species in freshwater ecosystems, with high conservation value (e.g. Vaughn and Hakenkamp 2001, Gutiérrez et al. 2003). To be able to preserve the mussel fauna worldwide, as well as their hosts, direct conservation strategies need to be established (Geist 2010). Therefore, knowledge about the interaction between individual mussel species and their habitats is of utmost importance, and particularly so the co-evolutionary link between mussels and fish hosts. Host fish assemblage and abundance strongly determine mussel abundance and community structure (Vaughn and Taylor 2000, Arvidsson et al. 2013). Thus, there is great need to evaluate the adaptive mechanisms behind the parasite-host interactions, something that hitherto only has been

4 understood for a few of the thousand freshwater mussel species existing worldwide (Strayer et al. 2004).

The objective of this introductory essay is to review the mussel – host-fish interactions of unionoid mussels, and relate these general interactions to the threatened thick-shelled river mussel Unio crassus (Philipsson 1788), the target species of my PhD thesis. Therefore, the species’ life history traits are discussed in a broad context of freshwater mussel ecology. As U. crassus is the most threatened European unionoid (Lundberg 2007), its status and threats are summed up to be able to relate to essential research and conservation of the species. So far, the question of the relationship between U. crassus and its hosts has only been addressed a few times, mostly in Germany, the Czech Republic and Luxemburg. Here, the European minnow (Phoxinus phoxinus), the European chub (Squalius cephalus) and the bullhead (Cottus gobio) have been identified as major host fish species (e.g. Bednarczuk 1986, Maaß 1987, Hochwald 1997, Thielen 2011, Douda et al. 2012, Taeubert et al. 2012a). However, it has also been shown that the host fish use varies between different drainage areas and their fish populations (e.g. Hochwald and Bauer 1990, Taeubert et al. 2012a). Wengström (2009), for example, reported on mussel population with successful recruitment where minnow, chub and bullhead were absent. This indicates a need for further research clarifying the relationship between mussels and fish for the entire mussel distribution area, so that one can establish conservation strategies for mussels and fish. My doctoral research will be presented at the end of this introductory essay.

2. Freshwater mussel ecology – the order Unionoida

In this chapter, I provide an overview of the general life cycle of freshwater mussels, along with specifications for each life cycle stage for U. crassus. First, I discuss sexual strategies and larval development which secondly is followed by a discussion of the mussel – host-fish relationship.

2.1. Sexual strategies and larval development Having derived from marine mussel species, all three freshwater mussel lineages (cyrenids, formerly the corbiculids, Bieler et al. 2010; dreissenids and unionids) developed their mode of reproduction from oviparous broadcast spawning towards ovoviviparity and even euviviparity. Thus, in contrast to their marine ancestors that cast out sperms and eggs separately in free flowing water resulting in external egg fertilization and development of planktonic veliger larvae, freshwater mussels adopted parental care (i.e. brooding). Accordingly, female mussels keep their eggs in the marsupium for fertilization by filtered sperms. Egg development in euviviparous mussel species, mostly sphaeriids (227 spp., 19% of the mussel species worldwide; order Veneroida), occurs in eggs poor in yolk where nutrients are provided over the maternal marsupium tissue (Korniushin and Glaubrecht 2003). Here, no larval stage can be found but “crawl-away juveniles”. Most unionoids (681

5 spp., 57%) represent ovoviviparous mussel species which brood their larvae in yolk rich eggs until transformed to larvae, which, unlike the marine veliger larvae are host fish dependent. Particularly in streams, the abolishment of the free-swimming larvae can be regarded as an evolutionary adaptation to lotic waters, where drift can be maladaptive (Graf 2013).

Compared to e.g. the sphaeriids that produce few offspring, the fertility of unionoids is generally high and probably a “result of the adoption of a parasitic life style” (Watters 2001). Thus, fertility depends on the species’ reproductive mode. Unionoid mussels that produce small larvae (e.g. Margaritifera margaritifera, the freshwater pearl mussel, FPM), for example, show higher fertility than mussels producing large larvae (e.g. Anodonta spp.), as energetic trade-offs do not allow for producing large and numerous larvae (Bauer 1994). Additionally, a general positive relation between fecundity and age/size has been found for many mussel species (Bauer 2001). However, food supply and temperature are important factors affecting fitness. The long-living and slowly growing headwater stream species FPM is generally highly fecund, but shows considerable variation in larval production for all age classes. This trait has also been observed for U. crassus, which usually occurs downstream of FPM habitats in watersheds, albeit U. crassus produces higher developed larvae before expulsion in free flowing water (Hochwald 1997). Variations in larval production may be explained by the level of fitness of each individual rather than by size/age relations (Bauer 1998). Furthermore, the degree of egg fertilization can fluctuate greatly. Hochwald and Bauer (1990) observed about 10% undeveloped eggs in gravid female U. crassus even at high mussel density. The authors explain this as natural failure rate during egg development, or because sperm concentration is below a critical level. Sperm provision is therefore, together with female fertility, an important parameter for successful mussel population recruitment.

Most unionoids are diocious, i.e. the sexes are separate (Kat 1983). Nevertheless, many species are believed to switch to hermaphroditism when population density is reduced, as e.g. the Australasian Hyriids (Walker et al. 2001), the FPM (Bauer 1987, but see Österling 2014) and the Anodonta species (Bauer 2001d) in Europe. Representatives of strictly diocious species are the North American Anodonta species A. corpulenta, A. gibbosa and A. wahlamatenis (Heard. 1975) and U. crassus in Europe, where the sex ratio is described as 1:1 (Hochwald and Bauer 1990). Generally, eggs are transported from the gonads via the oviduct, through the genital aperture into the cloaca to the gills where they get fertilized by sperm released by male mussels situated upstream of the females in lotic freshwaters (Coker et al. 1921).

One or both pairs of gills of female unionoids are used as breeding pouches, or marsupium, for eggs. In gravid mussels the marsupium appears swollen (Harms 1909). Margaritiferids hold the least developed demibranches (gill structures) among all mussel species worldwide as they brood their eggs in all four gills (tetragenous) and consequently have reduced filtration capacities (Bauer 2001c). Other species do not have this problem as they have their marsupium in either the outer (ectobranchious) or the inner (endobranchious) gills.

Anodonta and Unio species, to which U. crassus belong, use the ectobranchious breeding pouches whereas Etherioidea, occurring in the tropics of America and Africa, use the endobranchious demibraches. Partial use of inner or outer gills is found in the family Hyriidae and the genus Lampsilis which is regarded as advanced brooding strategy. Furthermore, it seems that many unionoid mussel species (except etherioids) can vary the amount of brood according to their level of fitness (Hochwald 2001). Thus, reduced filtration rate due to brooding activity generally causes a challenge for energetics which, however can be adjusted by regulating the extent of larval production.

There are two larval types within the order Unionoida, the lasidium and the glochidium. The lasidium type of larvae is found in the superfamily Etherioidea. Lasidia development to young mussels can either be similar to glochidia development where the egg develops to a larva metamorphosing to a young mussel in a host cyst, or can continue through a second larval stage, the “haustorium” which finally develops to a young mussel (Parodiz and Bonetto 1963). Additionally, some American mussel species show direct development from egg to young mussel without any larval stage (e.g. Anodonta inbecilis) (Wächter et al. 2001). The term “mussel larvae” will henceforth be used interchangeably with “glochidia”, while information on lasidia will be specified explicitly. The glochidia larval type is found in the superfamily Unionoidea (Bauer 2001a) which represents about 80% of the Unionoida (Barnhart et al. 2008), comprising three families, the Unionidae (the largest family), the Margaritiferidae and the Hyriidae (Strayer 2008). In Sweden, only the FPM (EN) belongs to the Margaritiferidae, whereas three Unio spp. (U. crassus (EN), U. tumidus and U. pictorum (NT)) and three Anodonta spp. (A. cygnea, A. anatina, Pseudanodonta complanata (NT)) belong to the family Unionidae (IUCN Red List Categories: EN, endangered; NT, near threatened; Bjelke et al. 2010). The distribution area of Hyriids is restricted to South America and Australasia (Bogan 2008).

Breeding strategies have been divided into long- (brachytictic) and short-term (tachtictic) breeders (Lefevre and Curtis 1910). Long-term breeders keep their larvae in their marsupium from fall until spring the year after (e.g. Anodonta imbecilis, Heard 1975; A. cygnea, A. anatina, Harms 1909). This leads to relatively large (~400µm) and morphologically well- equipped triangular shaped larvae carrying a larval thread (Wächter 2001). In contrast, short-term breeders release their small (~50µm) and less developed spherical glochidia a few weeks after fertilization (e.g. the FPM, Wächter et al. 2001; Anodonta gibbosa (Heard 1975). Interestingly, U. crassus lies in between those two breeding strategies, with egg fertilization in spring and release of medium-sized glochidia (~200µm) during late spring and summer (Bednarczuk 1986). U. crassus is nevertheless regarded as a short-term breeder and is capable of reproducing up to five times per year (Hochwald 2001) which has not been observed in other European species. Interestingly, reduced fertility has been found for older U. crassus females at later spawning events (Hochwald 1997). From an evolutionary perspective, this indicates that U. crassus has developed a reproduction strategy where

7 larvae are spread over extended time periods, potentially to extend the time span for larvae to encounter suitable host fish.

In summary, freshwater mussels evolved different sexual strategies and larval developments, where they have maximized offspring number with the smallest energetic cost. Which reproductive strategy the different mussel species adapted over time depends on the freshwater habitat that was colonized. The imposed selection pressure after the invasion of marine species to freshwaters, however, led to a shared adaptation of a parasitic life stage on fish within the order Unionoida (Graf 2013). Despite this common adaptation, individual interactions between fish and mussels depend on fish community structure, fish behaviour and habitat and have led to a variety of interactions between mussel species and their hosts.

2.2. The mussel – host-fish relationship Mostly, unionoid mussel larvae (glochidia) are obligate parasites on host fish. Without a suitable host, mussel larvae cannot develop and metamorphose into juvenile mussels. Consequently, glochidia die within a few days in the absence of hosts, depending on mussel species and temperature (Zimmerman and Neves 2002). According to Jansen et al. (2001) the highest glochidia mortality occurs after larval release from the adult mussel. This is also due to the fact that none of the larvae types are capable of locomotion (Lefevre and Curtis 1910), which decreases the likelihood of getting in contact with a suitable host. Also, this excludes selective host fish choice by the larvae (Bauer 2001b). Most mussel species compensate this dilemma by producing high numbers of larvae (Wächter et al. 2001) and/or reproducing several times per year, releasing their larvae asynchronously within the population, as is the case for U. crassus (Hochwald 1997). Hastie and Young (2003) state that larval release may be triggered by environmental cues, causing synchronized events in FPM populations. This timing may depend on temperature and follow an effect of thermal summation. However, fish presence and odours may also be used as cues to time larval release with host presence to increase infestation success (Jokela and Palokangas 1993). Thus, it seems that evolutionary adaptations of mussels include both high numbers of larvae, and larval release that is synchronised to host presence.

Host fish attraction by adult unionoid mussels is an evolutionary adaptation facilitating host encounter, serving to decrease glochidia mortality (Strayer et al. 2004). Examples of visual attraction include moving lures, which are modifications of the mantle margin, imitating fish or macroinvertebrates (e.g. Lampsilis spp.), or filamentous egg conglutinates mimicking worms. Moreover, transparent mucus structures, formed out of larvae, can look like small fish and lure foraging fish (Wächter 2001). The above host fish attraction strategies have not been described for European species. However, a so called spurting behaviour has been observed for the European species Pseudanodonta complanata (Hazay 1881) in Germany and U. crassus in Germany and Switzerland (Vicentini 2005; Beckmann 2007; Zettler and Jueg 2007), where the adult female mussel crawls to the stream edge, adjusting its posterior

8 end above the water level so that glochidia can be ejected up to one meter over the stream surface. Fish are probably attracted by the water splash, mistaking it for prey, and are infected upon foraging attempts. So far, spurting behaviour has not been seen for any Swedish freshwater mussel (personal communication Stefan Lundberg, Naturhistorska riksmuseet Stockholm 2013). Furthermore, glochidia attachment can be improved when larvae are equipped with an attachment thread. First, the thread reduces sinking rate of individual glochidia by 30%, and secondly it links together multiple larvae in a line. Thus, one larvae that attaches to a fish tow further larvae increasing infestation success (Jansen et al. 2001). Mussel species in lotic habitats are often threadless (e.g. M. margaritifera, U. crassus, P. complanata) whereas lentic species A. anatina, U. tumidus and U. pictorum carry threads (Wächter et al. 2001).

The position of attachment of mussel larvae on a fish is determined by extrinsic conditions such as water current and shear stress, as well as intrinsic conditions as fish behaviour and anatomy, but also larval morphology (Wächter et al. 2001). Hooked glochidia can attach to gill filaments and to external parts of the fish, particularly to opercula and fins (Arey 1932b). In spite of U. crassus glochidia having hooks, encapsulation of larvae fixed on the fish’s body surface is rarely successful, and it leads to higher mortality than when glochidia attach to the gills (Hochwald 1997, Engel 1990). Glochidia without hooks (e.g. FPM) are mainly gill parasites (Harms 1907a).

The encapsulation (interchangeably with encystment) of mussel larvae that attach to fish fins or rays has been described as wound closure, a reaction induced by lesion of blood vessels and performed by host epithelial cells (Arey 1932a). Encapsulation speed varies depending on mussel and fish species and fish condition, and has been estimated to take six hours for both FPM (Scharsack 1994) and Unio glochidia (Maaß 1987). Encapsulation can also occur if larvae attach to non-hosts, but here the cyst formation shows more irregularities than when larvae attach to suitable hosts. Also, the glochidia can be killed due to cellular defence, with subsequent expulsion within one day, after which a healing process starts immediately in the fish tissue (Arey 1932a, b). Attachment of a mussel larva on a suitable host mostly leads to encapsulation, enabling successful metamorphosis from glochidium to juvenile mussel. The process of juvenile release from the host fish after metamorphosis has yet to be fully clarified. Several authors propose that the juvenile mussel mechanically releases itself by pedal movement causing cyst rupture (e.g. Lefevre and Curtis 1910, Wächter et al. 2001), whereas Dodd et al. (2005) suggest that the host fish tissue retreat due to histolytic processes triggered by the juvenile. A search of the literature showed that there are few studies of the encapsulation, and particularly the release process of glochidia from fish tissue. A better understanding of the inner cellular substances participating in these processes could help explain why successful metamorphosis mostly occurs in fish gill tissue and fewer on fins, or why non-suitable hosts expel mussel larvae while suitable fish tolerate glochidia infestation over different periods of time.

Generally, the duration of the parasitic phase varies greatly depending on mussel species and breeding type (Bauer 2001c), but also on host fish species and quality and tissue type (Levine et al. 2012). Taeubert et al. (2013a), for example, measured slower metamorphosis when FPM larvae were attached to gill rakers than to gill filaments, which they ascribed to lower nutrition supply in gill-raker tissue. Moreover, many studies have shown that water temperature is an important parameter influencing the rate of metamorphosis (e.g. Harms 1907a, Lefevre and Curtis 1910). For U. crassus, Bednarczuk (1986) reported juvenile mussels after a 27-28 days parasitic stage on Squalius cephalus, Scardinius erythrophtalmus and Perca fluviatilis, without reference to water temperatures. The results of Hochwald and Bauer (1990), however, show successful metamorphosis on fish hosts Squalius cephalus, Phoxinus phoxinus and Cottus gobio after 28 days in 12 °C. At this temperature, U. crassus glochidia metamorphosed within 36-52 days in the experiment of Taeubert et al. (2013b), who tested the host Phoxinus phoxinus, at three temperatures. In their experiment, parasitism duration decreased with increasing temperature; metamorphosis occurred already after 11-15 days when fish were kept at 23°C. However, the lowest glochidial mortality was reported at 17 °C (16-28 days to metamorphosis) and consequently this temperature has been proposed as the optimum temperature for successful metamorphosis.

Whether the term “infection” defines the parasite-host interaction for freshwater mussels and their host fish correctly, has lately been discussed lively, mainly based on a lack of knowledge of the harm to hosts. However the word “infection” originally was used to describe a medical infection by bacteria, fungus or virus, “localised as in pharyngitis, or widespread as in sepsis, and are often accompanied by fever and increased number of white blood cells” as described by the American Heritage® Science Dictionary. As glochidial infestations are not infectious, researchers at a symposium (International Meeting on Biology and Conservation of Freshwater Bivalves in Bragança 2012) suggested substituting the term “infection rate” to “encystment rate” or “attachment rate” (anonymous reviewer). Important to mention is that fish mortality has only been observed when fish have been subjected to large numbers of glochidia under artificial laboratory conditions, i.e. not in nature (Lefevre and Curtis 1910, personal comment Österling 2014). However, evident harm to fish due to overinfestation under semi-natural conditions was found in a salmon farm located downstream of a FPM location in West Norway, where mussels occurred in high densities. In either case, larval metamorphosis success obviously requires both nutrients and energy, which, according to Jansen et al. (2001), suggests a negative effect of mussel larvae on fish hosts. Thomas et al. (2013) allude to a respiratory burden for fish when glochidia attach to gills, whereas no other serious physiological effects could be observed. However, glochidia are equipped with a mushroom body, formed by mantle cells, which are “the primary sites of intracellular digestion of the larval adductor muscle and host tissue during metamorphosis” (Fisher and Dimock 2002). This indicates that host tissue digestion takes place and cause harm to fish. Mechanical harm to hosts may also occur when glochidia grow on the fish gills, especially if they have a long parasitic stage, e.g. as with FPM growing from

70 to 400-500µm during nine months of parasitism (Bauer 2001c). This is not the case for U. crassus glochidia, as their pre-parasitic size is about 200µm and growth is only about 2.5 to 4.0% during 28 days before hatching from fish (Täubert 2012b). U. crassus is, on the other hand, equipped with hooks that may increase the physical harm to the host.

Fish can expel glochidia that are attached to fish tissue, a reaction that has been described as an immune response with antibody production against glochidial tissue (e.g. O’Connell and Neves 1999). This immune response depends on fish body condition at infestation (Frost 1943) and if the fish has been infested before (Jansen et al. 2001). Details about how this defence mechanism works from a physiological point of view are not well understood. There seems to be two types of immunity, natural and acquired immunity (Arey 1932a). Fish without natural immunity, or fish that have not yet acquired immunity, can be considered as suitable hosts. It is however still unclear how the immune response is linked to the encapsulation rate. For conservation purposes, it would be interesting to know if there is an optimal encapsulation rate that minimises the immune response and optimises glochidial transformation (Dodd et al. 2005). The results of Hochwald (1997), who investigated glochidial survival of U. crassus larvae on European minnows (P. phoxinus), indicate lower glochidial mortality when fish were highly infested. Similar results were obtained from the common rudd (Scardinius erythropthalmus) when the fish had previously been infested with glochidia from Anodonta anatina. This suggests that high infestation rates depress the immune system of fish. Furthermore, many studies suggest that older fish, which are more likely to have had a previous encounter with mussel larvae are less suitable for successful mussel metamorphosis even though they have large surface areas where larvae can attach (e.g. Arey 1932a, Bauer 1987, Blažek and Gelnar 2006, Strayer 2008). Hochwald (1997), however, did not find a difference in glochidia mortality rate when comparing minnows (P. phoxinus) that had been infested one or two times, nor was there an effect of fish size or age. This may indicate that P. phoxinus may be an ideal host for U. crassus, albeit U. crassus has more than one host. Taeubert et al. (2012a), for example, concluded that there are at least three popular hosts for U. crassus in the Danube drainage: P. phoxinus, the European chub (Squalius cephalus) and the European bullhead (Cottus gobio). Interestingly, there are additional fish species that act as hosts for U. crassus in other drainage systems (Tab 1).

Host fish mapping is a method to describe the geographic distribution of the mussel and its different host species. A complete host fish evaluation requires one to consider the endpoint of metamorphosis success i.e. the proof that juvenile mussels hatched from fish. Fish should therefore not only be caught in nature to investigate glochidia encapsulation rate, but should also be caught and transferred to laboratories where metamorphosed juveniles can be collected from natural and/or artificially infested fish (Taeubert et al. 2013c). Thus, observations of natural encystment rates on “ecological hosts” in mussel streams are equally important as lab experiments assessing “physiological hosts” (Levine et al. 2012).

Fish species Suitable Not suitable Alburnoides bipunctatus T(+) Alburnus alburnus W T(-) Acipenser ruthernus T(-) Tab. 1 Fish species tested Carassius carassius M as hosts for U. crassus and Chondrostoma nasus T(+) categorized suitable or not Cottus gobio T(-), H, LB suitable according to the Gasterosteus aculeatus T(-), E following authors: B, Gobio gobio M B Bednarczuk (1986); E, Engel Gymnocephalus cernua M (1990); H, Hochwald (1997); Leucaspius delineatus M HB, Hochwald and Bauer Leuciscus idus T(-) (1990); LB, Lundberg and Lota lota W Bergengren (2008); M, Neogobius melanostomus T(-) Maaß (1987); T, Taeubert Onchorynchus mykiss T(-), B et al. (2012a, b); W, Perca fluviatilis T(-), B, M Wengström (2009). (+) and Phoxinus phoxinus T(+), H, HB, M, LB (-) indicate higher and Pungitius pungitius E lower host quality Rhodeus serioeus T(-) according to Taeubert Rutilus rutilus B, W T(-), M 2012a, b. Salmo trutta E T(+), M, HB Scardinius erythrophythalmus T(+), M Squalius cephalus T(+), B, E, M Tinca tinca B, M

The number of fish species used varies depending on whether the mussel species is a host- fish generalist or a host-fish specialist (Strayer et al. 2004). Host generalists such as the North American mussel Cumberlandia monodonta, found in downstream parts of watersheds, can parasitize up to 36 fish species (Strayer 2008). Host specialised mussels, mostly occurring in headwater streams only use one or a few host species, e.g. the FPM using only brown trout (Salmo trutta) or Atlantic salmon (Salmo salar) as hosts. Host fish range also increases with mussel larvae size, independent of species, as large larvae often are equipped with hooks enhancing adherence on tissue, suggesting why e.g. Anodonta spp. with its relative large larvae use many hosts (Bauer 2001c). Non-host-specialised mussels mostly use co-occurring, local fish species independent of fish taxonomy (Barnhart et al. 2008). U. crassus seems to be a host generalist rather than a host specialist, but could not be accurately classified as a specialist or a generalist, as they parasitize on several but not all of the fish species in the assemblage (Hochwald 1997). However, to be able to understand why only certain fish species act as hosts, physiological and behavioural aspects of both the mussel and the fish needs to be taken into account.

Fish behaviour plays an important role for infestation success, as some fish species remain at mussel locations only during e.g. foraging, migration or spawning. The spawning season of the European minnow, for example, overlaps with the reproductive timing of U. crassus (Frost 1943). The benthic bullhead is also exposed to mussel glochidia, but seems comparably less vulnerable to infestation, probably since the opercula cover the gills more completely in bullheads. Bullheads may still act as important hosts for U. crassus (Lundberg et al. 2007) as they may occur at very high densities in small streams, particularly (Mills and Mann 1983). As bullheads have relatively small home ranges (Knaepkens et al. 2004), they probably lead to small-scale dispersal for parasitic larvae, whereas migratory hosts such as the European chub may transport mussel larvae to different habitats over extensive distances. Fish behaviours contribute to mussel infestation probability and dispersal.

The main dispersal mechanism for mussels is mediated by transportation of parasitic mussel larvae on host fish, and is even mentioned as the greatest benefit for mussel larvae in the parasite-host relationship (Barnhart et al. 2008). Generalist mussel species, with large host fish ranges, have potentially higher chances to be transported to different habitats, and migratory fish can spread juvenile mussels over long distances. Downstream drift by mussel larvae released from the maternal marsupium can also act as mussel dispersal, but the brief life span of expelled mussel larvae limits this as an efficient dispersal mechanism. Adult mussels can also disperse, by horizontal movement with up to 5m in 4 weeks for U. crassus, mostly directed upstream (Körner 1998-2001). Here, higher activity has been found for adult mussels 4 years of age in search of suitable habitats (Lang 1998). This adult movement should constitute a minor dispersal vector compared to that of glochidia on fish.

Adult mussels are also capable of vertical movement. During winter, mussels often stay completely buried in the sediment, or just underneath the bottom surface with only the siphons visible (Baumgärtner and Heitz 1995). Vertical mussel distribution has been described to occur in different layers with a tendency for larger and older mussels sitting near the surface, above smaller and younger mussels (Trudorancea and Gruia 1968). It is assumed that a sediment depth of 35-50cm can be reached by freshwater mussels, particularly by younger and thus smaller individuals (Jungbluth 1993). U. crassus has been found as low as 30cm below the sediment surface (Engel 1990). Temporal decreases in vertical movement activity can be seen during the reproductive season of adult FPM, when mussels can be found at the sediment surface. Particularly female mussels remain on top of the sediment with the siphons wide open. This facilitates the intake of free-flowing sperm needed for egg fertilization, as well as it enhances oxygen provision. In this way, female mussels get enough oxygen even though the filtration rate is reduced due to marsupial brooding (Harms 1907b).

Vertical movement occurs also in juvenile mussels when they migrate into the river sediment after having fallen off their host fish. A relatively long foot enables burying and high movement capacity. In the hyporheic interstitial zone, juveniles seek refuge from

predators such as fish or crayfish (Strayer 2008), and search for food such as detritus, algae and bacteria. During the early post-parasitic stage, juveniles pedal feed as their gills have not yet been developed (Wächter 2001). Therefore, cilia creating a water flow transporting food items to the digestion system cover the foot. Juvenile mussels do not emerge to the sediment surface until having fully developed into sexually mature filter feeders (Coker et al. 1921).

3. Status and threats to freshwater bivalves

Drastic habitat destruction and modification of freshwater ecosystems have followed man’s increased economic status and standard of living since the industrial revolution in the 18th and 19th centuries (Bogan 2008). Freshwaters are particularly adversely affected by major threats, including (I) destruction or degradation of habitat, (II) flow modification, (III) water pollution, (IV) overexploitation and (V) invasive species (Dudgeon et al. 2006). Among those, dam construction for hydroelectric power or mills represents one of the most serious threats to lotic freshwaters, as it disrupts the four-dimensional nature of running waters (Ward 1989), and their ecological connectivity in particular (e.g. Calles and Greenberg 2005, Mueller et al. 2011). Dams also cause changes to water levels and flow. Forestry including clear-cutting is a wide-spread threat to running waters (Österling et al. 2008, 2010) as it generates habitat degradation causing destabilization of bottoms, particularly in the riparian zone, where trees and floodplains are removed. Eutrophication of whole river systems often follows use of manure in high concentration, which cannot be buffered if floodplains are absent. Additionally, canalization increases the access of non-buffered pollutants to freshwaters, as well as erosion and fine sediment loads (Geist 2011), all contributing to a global decline of freshwater molluscs, including bivalves (Lydeard et al. 2004, Österling and Högberg 2014). According to Freyhof and Brooks (2011), many fish populations are also threatened due to the anthropogenic land-use change, particularly due to pollution and migration obstacles, conveying negative consequences for mussels with parasitic stages. Furthermore, the introduction of alien species, such as the Eurasian zebra mussel (Dreissena polymorpha) and the Asian clam (Corbicula fluminea), causes high competition between invasive and native mussel species, with potential for extinction (Ricciardi 1998, Yeager et al. 1999). Overexploitation of mussels caused huge population declines (Strayer at al. 2004) as a market for the mother of pearl and pearls started blooming in the 16th century (Strayer 2008). Here, decorative art and expensive pieces of jewellery were made out of the shiny mother of pearl and pearls produced by some mussel species as a dirt-encapsulation reaction (e.g. FPM) (Lozoya and Araujo 2011).

Prior to the 19th century, mussels (e.g. the Unio species) still occurred in such high population densities that the mussels were fed to pigs and chickens, and shells were used as fertiliser (Baumgärtner and Heitz 1995). Today, freshwater molluscs is the most imperilled

taxonomic group both in Europe (Cuttelod et al. 2011) and globally (Bogan 2008). The reasons for the mussels’ high sensitivity to anthropogenic habitat alterations are directly linked to their ecology, i.e. the mussels’ complex life history traits. Acute toxicity or dredging can negatively affects all individuals in a population. Additionally, mussel larval survival depends on availability of suitable host fish, and a lack of hosts can lead to mussel population decline. According to Haag and Warren (1998) the variability of fish community structure, combined with the host fish infestation strategy by mussels, can have a greater impact on mussel community structure than habitat structure and variability. They also conclude that mussels that are host specialists and lack fish-attraction strategies suffer most from low host densities, in contrast to generalist mussel species with the ability to attract hosts.

Juvenile mussels are highly sensitive to pollution, eutrophication and clogged sediments with low oxygen concentrations (Jungbluth 1993). As juveniles are not able to choose their habitat when hatching from hosts, they are dependent on overall good sediment quality (Nagel and Badino 2001). High fine sediment loads in freshwaters due to e.g. agriculture, forestry, canalization or damming can cause juvenile mortality (Österling et al. 2010). Investigating recruitment failure for U. crassus, Hochwald and Bauer (1990) argue that high values of nitrate that can convert to nitrite at anoxic conditions is toxic for juveniles. Therefore, different nitrate thresholds have been prescribed for juvenile survival (Engel 1990, Hochwald and Bauer 1990), but still need to be confirmed in detail (e.g. Denic et al. 2013, Strayer 2012, 2014). Hence, the postparasitic juvenile phase may be a very critical stage in the mussels’ life cycle (Jansen et al. 2001).

Another local threat for unionoid mussels is the muskrat (Ondatra zibethicus) predation. Owen et al. (2006) studied the impact of this semi-aquatic rodent, native to North America, on mussel community. They concluded that muskrats negatively affect species composition, population size and age structure as it is mussel size and shape selective. However, compared to southern and central Europe, where the muskrat has spread rapidly since the early 20th century causing drastic mussel population declines, the muskrat in North America and western Europe has natural predators such as mink (Mustela vison), bob cat (Felis rufus), red fox (Vulpes vulpes) or coyote (Canis latrans), controlling the muskrat populations. In these regions, the muskrat has also been hunted for its fur. In Europe, U. crassus is the muskrats’ target mussel species, compared to e.g. Anodonta anatina and Unio pictorum (Zahner-Maike and Hanson 2001). Even though the muskrat does not eat every individual of a population, it may severely reduce mussel density. Together with other threats mentioned, this can lead to allee effects in sparse populations.

Allee effects include elevated extinction probability below threshold or critical population sizes (Vaughn 2012). As soon as the population size of mussels decreases, factors regulating successful recruitment can be adversely affected. This may be impoverished sperm availability in male-reduced populations as well as sperm dilution effects from low

population densities leading to great distances between males and females (Strayer 2008). To counteract allee effects, mussels may aggregate, the two sexes mate and hermaphroditism becomes common in some species (e.g. FPM). Therefore, some authors (cited in Strayer 2008) claim that a limited population size does not affect reproduction due to lack of sperm i.e. limited fertilization. However, Hochwald and Bauer (1990) did not observe hermaphroditism or that males and females approached in U. crassus populations and therefore suggest that U. crassus populations suffer severe extinction risk when the population size falls under a certain threshold. Here, a danger of inbreeding occurs in small populations suffering under a component allee effect that occurs when there is a positive relationship between any measureable component of individual fitness and population size or density (Berec et al. 2006). Thus, conservation programs for endangered mussel species focusing on breeding and culturing of mussels should carefully take into account inter- and intra-population genetic patterns and “maintain the genetic identity of evolutionary significant units (ESUs) and conservation units (CUs)” (Geist 2011).

4. Conservation strategies

Ecological knowledge about mussel – host-fish interactions is of crucial importance when working towards conservation of large freshwater mussels (Taeubert et al. 2012a). Host-fish mapping should take into account research on the fish immune reaction due to glochidia infestation and co-adaptation between mussels and fish (O'Connell and Neves 1999, Jansen et al. 2001, Dodd et al. 2005). In other words, research on the relationship between mussels and their host fish may help to adjust stream restoration incentives according to species habitat requirements (Zale and Neves 1982), including removal of migratory obstacles for fish (Vaughn and Taylor 2000). Additionally, re-meandering of channelized streams including re-establishment of floodplains is, for example, a suitable method to improve habitat quality for a variety of freshwater organisms as it can contribute to reduced erosion, stabilization of sediments, buffering of nutrient loads from agriculture and diversification of in-stream flow regimes, to mention a few possible positive effects (Roni and Beechie eds. 2012). Here, specific parameters as e.g. substrate heterogeneity or flow regime can be adjusted to meet the needs of the mussel and host fish.

Re-introduction of mussel species in streams where populations have been extinct or where mussel populations are decreasing can be performed by stocking of infested host fish (Zettler and Jueg 2007). Juvenile hatching rates from infested host fish as well as mussel survival are crucial parameters for evaluation of re-introduction efficiency. Suitable monitoring methods are needed, and the small size of juveniles after metamorphosis, around 200-500µm, poses serious challenges to developing such a monitoring program. Often, one needs to wait several years before being able to evaluate the outcome of a re-

introduction by estimating the number of mussels emerging from the interstitial sediment to the riverbed surface. As research and conservation projects rarely have time to wait so long, captive breeding programs have been established (Lefevre and Curtis 1912). Here, juvenile mussels, developed on artificially infested hosts, are hatched and reared in the laboratory until the mussels reach an age and size that can be easily spotted in rivers. This has been conducted successfully for many North American (e.g. Barnhart 2005) and two European species (reviewed by McIvor and Aldridge 2008), and so far once for U. crassus in Luxembourg (Eybe and Thielen 2010). The reared mussels are usually transferred to watersheds, after marking them in the lab to enable identification in nature and evaluating re-capture success. Marking methods like waterproof colour marking (Hochwald 1997), engraving (Barnhart 2003), and PIT (passive integrated transponders) tagging (Hua et al. 2012) have been used. Recent development also includes fluorescent marking of juvenile mussels. However, this technique needs to be improved as low survival prevails (Lavictoire 2012). Engel (1990) conducted a survival study using mesh-covered hole plates, later known as “Buddensiek cages” (Buddensiek 1995), which were attached to tree roots slightly above the stream bottom. Thus, instead of marking juveniles, the small mussels could be re- captured as they were held in individual compartments. Unfortunately, juvenile survival rate was greatly reduced due to clogging of the mesh, in spite of repeated cleaning. In other words, techniques for juvenile re-capture are needed as they would tremendously promote research on juvenile mussel ecology, e.g. habitat requirement, foraging and survival. Furthermore, research is essential for mussel conservation as mussel propagation programs can be created more cost-effective focusing on juvenile re-introduction and reducing the effort in captive breeding.

5. My doctoral research

The aim of my dissertation is to investigate mussel-host interactions for U. crassus, considering the complex mussel life history traits. Few studies have hitherto been conducted on U. crassus, particularly in Swedish rivers, in spite of that, the need to preserve this national and pan-European endangered species is urgent, approved and prioritized. To facilitate the development and establishment of conservation programs, I address the following questions: (1) Which host-fish species are used by U. crassus in Sweden? (2) When does U. crassus reproduce, and are timing and encapsulation rates linked to fish host size and/or fish/mussel densities? (3) Is glochidia release by female U. crassus dependent on temperature and host fish presence? (4) Does host fish suitability differ between streams and does co-adaptation influence the juvenile metamorphosis success? (5) How high is juvenile survival when re-introduced in natural habitats?

1. Identification of fish species used as hosts by U. crassus is an essential part of the studies of mussel-host interactions and improves conservation actions for mussels and host fish.

Hence, fishing will be conducted within the species distribution area in Sweden along a north-south gradient. Seven mussel streams located in the Region of Skåne, Blekinge, Jönköping, Östergotland and Södermanland will be sampled during the reproduction season of U. crassus. Preserved fish will be examined for glochidial infestation in the laboratory wheareas ITS rDNA analyses of single glochidia will help identifying U. crassus and separating the species from other co-occurring mussel species in the streams. This study will be combined with investigations of excystment of juvenile mussels from naturally infested fish enabling evaluation of fish species suitability as “ecological hosts”.

2. Assessment of the reproductive period of U. crassus will help us to understand the link between changes in temperature, host fish presence and host infestation with mussel spawning events. My investigations will therefore include comparisons of the proportions of gravid mussels, their size and repeated spawning across mussel densities. Host quality will be evaluated by means of natural encystment rates on local host fish species electro-fished which will be referred to both host and mussel density. Hypothetically, the temporal dynamics of natural infestations should respond to gravidity dynamics. Moreover, fish host size-dependent capacity to carry glochidia will be investigated.

3. Water temperature is a factor known to have an impact on the release of glochidia larvae. An ability to sense the presence of host-fish may also be a mechanism that triggers larval release of female mussels and result in high infestation. In my third study, I address the question if larval release of gravid U. crassus females is affected by host-fish presence and water temperature, or a combination of both. I will investigate the timing of glochidia release from gravid mussels in treatments with and without fish presence at three water temperature levels simulating cold, intermediate and warm temperatures at the early reproduction season of U. crassus.

4. The relationship between mussels and fish will additionally be investigated by means of artificial infestation of fish under standardized aquarium experimental conditions. Here, I question if U. crassus is a host generalist or specialist and if host fish species suitability varies between drainage areas. During these laboratory studies the infestation rate of mussel larvae on fish will be followed over the parasitic stage and the juvenile metamorphosis success quantified which enables evaluation of host functionality. Also, by investigating infestation and survival rates of mussels on sympatric and allopatric host fish species, common garden experiments will aim to evaluate if mussels in separate catchments have adapted locally to catchment-specific fish host composition. Moreover, fish that are infested with mussels under known, laboratory conditions and infestation rates will be evaluated for re-infestation rates, where possible immune responses in fish can reduce infestation rates in a second infestation round.

5. Studies of habitat requirements of early mussel life cycle stages such as fertilized larvae on adult mussels, encysted larvae on host fish and free-living juvenile mussels are

essential prerequisites for conservation programs focusing on mussel re-introduction. Therefore, I will conduct research in two habitat restored rivers where U. crassus shall be re-introduced. Gravidity of re-introduced adult mussels with origin in mussel streams will be investigated by means of standardized methods. Compatibility between the parasitic stage of U. crassus and potential host fish species from the two rivers will be evaluated by means of artificial infestation of fish under standardized experimental conditions. Juveniles hatched in these laboratory experiments will be re-introduced into the restored rivers and monitored for survival and growth. Survival and growth of juvenile mussels introduced in their native rivers, which have recruitment of juvenile mussels, will serve as controls.

6. Acknowledgements

My PhD research is funded by the European Commissions’ LIFE program fund and FORTUM. I would like to thank Ivan Olsson for initiating and managing our project UnioCrassusforLIFE. For more information, please visit: www.ucforlife.se.

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Introductory papers in Biology, Karlstad University:

1. Olsson, I.C. 2005. Migration av öring (Salmo trutta L.) 2. Calles, O. 2005. Fiskars migration och reproduktion i reglerade vatten – restaureringsåtgärder och dess effekter 3. Österling, M. 2005. Begränsande faktorer för utbredning och överlevnad av flodpärlmusslan (Margaritifera margaritifera L.) 4. Olsson, M. 2007. Infrastukturens effekter och betydelse av faunapassager 5. Gustafsson, P. 2007. Kantzonsvegetations inverkan på öring (Salmo trutta L.) i rinnande vatten 6. Cassing, G. 2009. Löträd, landskap och viltbete – en introduktion till studier av lövträdrekrytering och viltbete i olika rumsliga skalor 7. Enefalk, Å. 2012. Brown trout responses to woody debris in boreal streams 8. Watz, J. 2012. The foraging behaviour of steam salmonids during winter: The effects of temperature, light intensity, and surface ice 9. Nyqvist, D. 2013. Atlantic salmon kelts – repeat spawning and downstream migration 10. Lans, L. 2013. Beteende och metabolism: Hur ämnesomsättning och beteende påverkar viljan att migrera hos Atlantlax (Salmo salar) och öring (Salmo trutta) 11. Schneider, L. D. 2014. Ecology of the threatened thick-shelled river mussel Unio crassus (Philipsson 1788) with focus on mussel-host interactions